Method of identifying compounds that alter bag-1 mediated down-regulation of glucocorticoid-receptor transactivation

ABSTRACT

The present invention relates to new methods of identifying compounds that inhibit or reduce bag-1 mediated down-regulation of gluocorticoid-receptor (GR) tansactivation. These methods rely on the surprising findings, that first, a (poly)peptide comprising the motif X-Lys-Lys-Lys-Y-Arg-Arg-Arg also present is the cochaperone bag-1 is sufficient for DNA binding of this cochaperone and, second, this motif but not the E 2 X 4  domain of bag-1 is required for inhibition or reduction of FR transactivation. The present invention also relates to methods of refining the compounds identified with the above method as well as to methods of producing pharmaceutical compositions wherein compounds identified or compounds refined by the above-recited methods of the invention are formulated with a pharmaceutically acceptable carrier or diluent.

The present invention relates to new methods of identifying compoundsthat inhibit or reduce bag-1 mediated downregulation ofglucocorticoid-receptor (GR) transactivation. These methods rely on thesurprising findings, that first, a (poly)peptide comprising the motifX-Lys-Lys-Lys-Y-Arg-Arg-Arg also present in the cochaperone bag-1 issufficient for DNA binding of this cochaperone and, second, this motifbut not the E₂X₄ domain of bag-1 is required for inhibition or reductionof GR-mediated transactivation. The present invention also relates tomethods of refining the compounds identified with the above method aswell as to methods of producing pharmaceutical compositions whereincompounds identified or compounds refined by the above-recited methodsof the invention are formulated with a pharmaceutically acceptablecarrier or diluent.

A number of documents including manufacturers' manuals is cited in thisspecification. The disclosure content of all these documents is herewithincorporated by reference.

BAG-1 (also denoted bag-1 throughout this application) was originallyidentified as an associating factor of the antiapoptotic factor bcl2(Takayama et al., 1995) and, independently, as a protein called RAP46that associates with the glucocorticoid receptor (GR) and other steroidhormone receptors (Zeiner and Gehring, 1995). It also is termed “hap46”,hsp70- and hsc70-associating protein (Gebauer et al., 1998). BAG-1 isinvolved not only in apoptosis and tumorigenesis (Thress et al., 2001;Turner et al., 2001), but also in the function of nuclear receptors. Forexample, BAG-1 enhances the transcriptional activity of the androgenreceptor (Froesch et al., 1998; Knee et al., 2001), while in the case ofthe vitamin D receptor evidence has been provided for both enhancement(Guzey et al., 2000) and inhibition (Witcher et al., 2001) oftranscriptional activity. In addition, BAG-1 interacts with the retinoicacid receptor (RAR) and inhibits its binding to retinoic acid responseelements on DNA as well as RAR-dependent transcription (Liu et al.,1998). Likewise, BAG-1 has been described as a negative regulator of GR,since it binds to the hinge region of GR and inhibits DNA binding andtransactivation of the receptor (Kullmann et al., 1998).

Before activation by hormone, GR resides in the cytoplasm, where itinteracts with various molecular chaperones in a step-wise fashion toattain the competent hormone binding state (Bresnick et al., 1989; Prattand Toft, 1997; Buchner, 1999). Central to this folding process are heatshock protein (hsp) 90, hsp70, and hsp70/hsp90 organizing protein (hop)(Dittmar et al., 1997; Pratt and Dittmar, 1998; Toft, 1998), whichbridges hsp70 and hsp90 via its tetratricopeptide repeat (TPR) domains(Scheufler et al., 2000). Hsp90, hsp70 and hop, possibly with theinvolvement of hsp40 (Dittmar et al., 1998), form an intermediatecomplex with GR (Dittmar et al., 1996; Pratt and Toft, 1997), from whichhsp70 and hop presumably dissociate to allow entry of p23 and one of theimmunophilins to the final complex, where GR gains competence of bindingto hormone.

The chaperone activity of hsp70 is modulated not only by BAG-1, but alsoby hsp40, C-terminus of hsp70 interacting protein (CHIP), and hsp70interacting protein (hip). Hsp40 enhances the ATPase activity of hsp70in vitro (Freeman et al., 1995) and the hsp70-dependent refolding inmammalian cells (Michels et al., 1999; Michels et al., 1997). Hip alsohas been identified as a positive regulator of hsp70 chaperone activity(Höhfeld et al., 1995; Nollen et al., 2000b). In contrast, CHIP has beenfound to inhibit the ATPase activity of hsp70 and to interfere withstable hsp70-substrate complexes (Ballinger et al., 1999). All isoformsof BAG-1, i.e. BAG-1L, BAG-1M, and BAG-1S, which derive from differenttranslation initiation sites localized on the same gene (Packham et al.,1997; Takayama et al., 1998; Yang et al., 1998), have been described inseveral studies to inhibit hsp70-dependent refolding activity in vitroand in vivo (Bimston et al., 1998; Zeiner et al., 1997; Höhfeld andJentsch, 1997; Nollen et al., 2000a; Nollen et al., 2000b). Moreover,BAG-1 was found to compete with the stimulatory action of hip (Nollen etal., 2000b). Hip, in turn, opposes the negative effect of BAG-1 onsteroid binding of GR (Kanelakis et al., 2000). The negative effect ofBAG-1 on steroid binding, however, was not observed by others(Schneikert et al., 1999).

While all these reports strongly suggest a cytosolic effect of BAG-1 onGR folding and activity, there are also data supporting a nuclearfunction of BAG-1. For example, BAG-1 has been reported to bindnon-specifically to DNA and to stimulate DNA transcription (Zeiner etal., 1999). Deletion of or mutations within the N-terminal ten aminoacids of BAG-1 abolish its DNA binding (Zeiner et al., 1999). Inaddition, it has been shown that BAG-1 is transported into the nucleusupon steroid binding and nuclear translocation of GR (Schneikert et al.,1999). This nuclear translocation is dependent on the C-terminal hsp70binding domain of BAG-1. Moreover, the inhibiting effect of BAG-1 on DNAbinding of GR (Kullmann et al., 1998) can be overcome in a cell freesystem by supplementing with increasing amounts of hsp70 (Schneikert etal., 2000).

While an hsp70 interaction domain of BAG-1 has been identified andcharacterised by crystallography in the C-terminal region (Briknarova etal., 2001; Sondermann et al., 2001), the function of the N-terminal partseems less clear. It has been suggested that a serine- andthreonine-rich E₂X₄ domain is necessary for the inhibitory function ofBAG-1 (Schneikert et al., 1999). If this indeed were the case, thecontrol of bag-1 mediated inhibition of GR transactivation would berather difficult to achieve due to the expected complex interplay of thevarious control elements involved. On the other hand, means and methodsfor the specific inhibition of bag-1 mediated downregulation of GRtransactivation are clearly desired. For example, such means and methodswould allow a reduction in the amount of glucocorticoid administeredduring glucocorticoid therapy. The main advantage of such an approach isthe reduction of adverse side effects triggered by glucocorticoids.

Thus, the technical problem underlying the present invention was toprovide such means and methods that will allow a specific inhibition ofbag-1 mediated suppression of GR transactivation with a view to reducethe adverse side effects in glucocorticoid-related therapy.

The solution to said technical problem is achieved by providing theembodiments characterized in the claims.

Accordingly, the present invention relates to a method of identifying acompound that inhibits or reduces bag-1 (for a sequence, see FIG. 6)mediated downregulation of glucocorticoid-receptor (GR) transactivationsaid method comprising the steps of (a) contacting a test compound or aplurality of test compounds with (aa) a (poly)peptide comprising themotif X-Lys-Lys-Lys-Y-Arg-Arg-Arg wherein X and Y represent 0, 1, 2, 3,4, 5 or 6 amino acids and wherein said amino acids allow binding of themotif to double-stranded DNA; and (ab) a double-stranded DNA underconditions that allow binding of said (poly)peptide to said DNA in theabsence of said test compound or said plurality of test compounds; andassessing whether binding of said (poly)peptide to said DNA occurs inthe presence of said test compound or said plurality of test compoundswherein inhibition or reduction of binding is indicative of the testcompound(s) being suitable to inhibit or reduce bag-1 mediateddownregulation of GR transactivation (including transactivation of allGR isoforms throughout this specification, preferably GRαtransactivation; for a specific sequence, see FIG. 7; however, alsoincluding GRβ transactivation; for a specific sequence, see FIG. 8).

The term “transactivation” means in accordance with the presentinvention enhancement of the rate of transcription from a gene promoterby a transactivator, for example, GR. In accordance with the presentinvention the terms “glucocorticoid-receptor transactivation” or “GRtransactivation” define the same phenomenon as“glucocorticoid-receptor-mediated transactivation” or “GR-mediatedtransactivation” and are used herein as synonyms.

The term “plurality of test compounds” is intended to mean, inaccordance with the present invention, at least two test compounds suchas 3, 4, 5, 6, 7, 8, 10 or 20, 50, 100, 200, 500 or 1000 or more testcompounds. The number of test compounds comprised in such plurality oftest compounds may be significantly higher such as amounting to 10⁴,10⁵, 10⁶ or more different compounds which may, for example, derive froma library of cDNAs or a collection of small molecules.

The term “(poly)peptide” denotes both peptides and polypeptides(proteins) wherein, according to a common understanding in the art, thelength of peptides is limited to molecules up to about 30 amino acids.

The term “comprising” has, in accordance with the present invention twodifferent meanings: The first meaning is “consisting of”. For example,in connection with the recited motif the term comprising would then meanthat the (poly)peptide consists of the modif. The second meaning denotesa surplus over the actually cited technical feature. In the example ofthe motif, additional amino acids N- or C-terminally thereof would beenvisaged.

As regards the above recited motif, it is understood that it comprisestwo variables X and Y as well as the trimeric lysine and argininerepeats. Both X and Y may represent between 0 and 6 amino acids.Advantageous embodiments are particularly those wherein Y representsbetween 0 and 3 amino acids. If X and/or Y represent more than 1 aminoacid, these amino acids may be the same or different. X preferablyrepresents one amino acid. It may also be bound to a complete proteinthat forms a fusion protein together with the (poly)peptide comprisingthe remainder of the motif. In any case, it is required that thecapability of the motif to bind to double-stranded DNA is retained. Inthis regard and in accordance with the present invention, inclusion ofthe E₂X₄ motif, as suggested by the prior art, into the (poly)peptide isnot required. Retainment of binding is easily ascertained. For example,binding to any DNA sequence can be assessed by the Biacore technique,electrophoretic mobility shift assays, filter binding assay, or bybinding of DNA to immobilzed protein or vice versa.

The term “suitable to inhibit [. . . ] bag-1 mediated downregulation ofGR transactivation” indicates that downregulation is inhibited to 100%or close to 100%. The term .“suitable to [. . . ] reduce bag-1 mediateddownregulation of GR transactivation” indicates that downregulation isreduced at least 30% preferably at least 50% and more preferred at least70%. If a close to 100% or a 100% reduction is achieved, the meanings ofthe term “reduction” and “inhibition” overlap or are identical.

In accordance with the method of the present invention, the contactingof the test compound(s) with the (poly)peptide and the double-strandedDNA molecule (which comprises preferably at least 23 nucleotides perstrand) is effected under conditions that allow binding of said(poly)peptide to said DNA in the absence of said test compound. Thevarious compounds contacted may be put together at the same time, oneafter the other wherein a certain order is not required or two at atime. The same holds true for the further embodiments of the inventiondescribed below wherein also more than two compounds may be contacted ata time and the other compounds, as above, earlier as later.

Suitable conditions are determined by the skilled artisan withoutfurther ado. For example, testing may be effected under physiologicalconditions. Buffers that may be employed to ascertain such physiologicalconditions include phosphate buffers having a pH value of about 7.0. Anytechnique that allows determination of binding to any DNA sequence, e.g.the Biacore technique, electrophoretic mobility shift assays, or bybinding of DNA to immobilzed protein or vice versa, can be used toverify DNA binding under physiological conditions, e.g. phosphatebuffered saline with a pH of 7.0-7.5 and 150 mM salt (NaCl+KCl).

If a plurality of test compounds is contacted with said (poly)peptideand said double-stranded DNA and inhibition of binding is observed thisusually means that one or a few members of the plurality of compoundswill be causative for said inhibition. In other terms, said compoundwill bind to the DNA-binding motif of bag-1 and thus interfere withbinding of bag-1 to DNA.

It has been surprisingly found in accordance with the present inventionthat deletion of the entire E₂X₄ motif domain does not affect theability of BAG-1 to counter GR-dependent transcription. Furthersurprisingly, it was discovered in accordance with the present inventionthat a small DNA binding domain comprising the above-recited motif isnecessary for inhibition of GR function. Specifically, the first eightamino acids of BAG-1 or a motif related thereto and recited herein aboveare/is required for the inhibitory effect of BAG-1 on GR, while the E₂X₄domain is dispensable. The observations made in accordance with theinvention are particularly surprising since the art described a generaltranscription enhancing capability to the bag-1 related motif (Zeiner etal. 1999) which contrasts the inhibition of GR transactivation observedhere. Moreover, the binding of BAG-1 to DNA is due to the positiveelectric charge at the N-terminus. Mutations that inhibit binding to DNAcan not be functionally rescued by overexpressing BAG-1 with a pointmutation abolishing its interaction with hsp70, indicating that the DNAbinding and the hsp70 interaction domain must be present in cis.

This DNA binding domain is uncommon for transcriptional regulatoryproteins, because DNA binding appears to be non-specific (Zeiner et al.,1999). The non-specificity of DNA binding is in line with theobservation made in accordance with the invention that spacing of thetwo positively charged stretches of three lysines and three arginines isnot important for DNA binding. This suggests an interaction with DNA viaionic interactions of these positive charges with the negatively chargedphosphate backbone of DNA.

In accordance with the above, identification of a compound that inhibitsthe binding of the recited motif to said double-stranded DNA isimmediately indicative of this compound being useful as an inhibitor ofbag-1 mediated down-regulation of GR transactivation.

Whereas the applicant does not wish to be bound by any scientifictheory, the following model is proposed to account for the effects (FIG.5): BAG-1 is transported into the nucleus along with GR upon activationof the receptor with hormone (Schneikert et al., 2000). GR interactswith the chromatin at glucocorticoid response elements. The first stepin GR-mediated transactivation presumably is remodelling of the localchromatin structure by cofactors recruited by GR (Freedman, 1999). Oncethe chromatin is restructured, BAG-1 gains access to the DNA, whichinterfers with the further functions of GR for efficienttransactivation. Access of BAG-1 to the DNA might be promoted by therapid exchange with regulatory sites of GR (McNally et al., 2000).

BAG-1 would not be the only non-specifically DNA binding factor, whichbinds to specific places on the chromosome by virtue of associating withother factors. Another example is cdc6, an essential protein in yeastwhich is recruited to yeast replication origins in G1 by anotherreplication factor, origin recognition complex protein 1 (Wang, Feng, etal. 1999). It is intriguing not only that cdc6 alone bindsnon-specifically to DNA, but also that site-directed mutagenesisidentified the basic protein motif KRKK as essential for DNA binding andfunction of the protein (Feng, Wang, et al. 2000). Apparently, thismotif is very similar to the basic motif of the N-terminus of BAG-1,which is demonstrated in accordance with the present invention to be notonly essential for binding to DNA, but also for functional integritywith respect to the inhibition of GR-dependent transcription.

The method of the invention allows for the identification of compoundsthat will immediately (if formulated as a drug) or eventually (if usedas a lead compound) give rise to medicaments that are useful in thereduction of adverse side effects caused by glucocorticoid therapy.Specifically, the effects desired by the glucocorticoid therapy may beretained in quantitatively undiminished form whereas the adverse sideeffects are significantly reduced, if a medicament developed on thebasis of the present invention is administered in conjunction with theglucocorticoids. Further, the compounds may be effectively used in thetherapy of tumors, in the generation or progression of which activatedandrogen receptor or estrogen receptor plays a role. Thus, it has beenshown that the androgen receptor is upregulated by bag-1 (Knee et al.2001). In addition, it has been shown that higher levels of bag-1 arepositively correlated with enhanced survival rates of breast cancer. Inso far, breast cancer is an embodiment of the tumors mentioned above.Additionally, in further approaches the compound may replaced by theglucocorticoid in therapy.

In a preferred embodiment of the method of the invention, said motifrecited in (aa) is located at the N-terminus of said (poly)peptide.

In this embodiment, the location of the motif within the (poly)peptideis identical to its location within bag-1. This embodiment allows aparticularly convenient identification of suitable compounds due to itsreliance on the naturally occurring situation.

In another preferred embodiment of the method of the invention, said DNAcarries a readout-system that is activated by the binding of said(poly)peptide to said DNA.

The term “read-out system” means an experimental set up that allows easydetection (“read-out”) of a biological parameter of interest. Forexample, when electrophoretic mobility shift assays are used todetermine binding of peptides or proteins to DNA, the biologicalparameter of interest would be DNA binding and the read out would be theshifted radioactive bands on an acrylamide gel, as detected beautoradiography or an imaging system. The experimental set up wouldinclude incubation of protein and (labelled) DNA in a suitable buffer,loading on and electrophoresis through an acrylamide gel followed byautoradiography.

Preferably, by said read-out system the modulation of a signal ismeasured in the presence or absence of said compound. Thus, themodulation of the measured signal indicates whether said compound issuitable for the downregulation of GR-mediated transactivation.

In accordance with this preferred embodiment of the method, binding andconsequently identification of compounds is rendered particularly easy.In particular, the read-out system indicates whether a prima faciesuitable compound has prevented binding of bag-1 to the DNA or not. Inorder to eliminate false positive signals, further tests may benecessary. Employing such tests is within the common knowledge of theperson skilled in the art. Further embodiments of such read-out systemsare described in the appended examples.

In addition, the present invention relates a method of identifying acompound that inhibits or reduces bag-1 mediated downregulation ofglucocorticoid-receptor transactivation said method comprising the stepsof (a) contacting a test compound or a plurality of test compounds with(aa) a (poly)peptide comprising the motif of X-Lys-Lys-Lys-Y-Arg-Arg-Argpreferably at its N-terminus wherein X and Y represent 0, 1, 2, 3, 4, 5or 6 amino acids; and a domain that is the hsp70 binding domain of bag-1or functionally equivalent; (ab) polypeptide representing the GR or afunctionally equivalent molecule; and (ac) double-stranded DNA moleculecomprising a binding site for the GR under conditions that allow theformation of a functional (poly)peptide complex consisting of said(poly)peptides recited in (aa) to (ab) and binding of said polypeptiderecited in (ab) with said double-stranded DNA molecule in the absence ofsaid test compound or said plurality of test compounds; and (ba)assessing whether said polypeptide recited in (ab) binds to saiddouble-stranded DNA wherein an increase of binding is indicative of thetest compound(s) being suitable to inhibit or reduce bag-1 mediateddownregulation of GR transactivation; or (bb) assessing whether saidcomplex formation and/or DNA-binding recited in (ac) results in atransactivation of GR wherein an increased level of transactivation isindicative of the test compound(s) being suitable to inhibit or reducebag-1 mediated downregulation of GR transactivation.

As regards the above recited motif, it is understood, as already definedfor the embodiments above, that it comprises two variables X and Y aswell as the trimeric lysine and arginine repeats. Both X and Y mayrepresent between 0 and 6 amino acids. Advantageous embodiments areparticularly those wherein Y represents between 0 and 3 amino acids. IfX and/or Y represent more than 1 amino acid, these amino acids may bethe same or different. X preferably represents one amino acid. It mayalso be bound to a complete protein that forms a fusion protein togetherwith the (poly)peptide comprising the remainder of the motif. In anycase, it is required that the capability of the motif to bind todouble-stranded DNA is retained.

The terms “increase of binding” and “increased level of transactivation”refer to increases of at least 25%, preferably at least 50%, morepreferred at least 75% and most preferred at least 100% of the initiallevel.

This embodiment of the present invention does not rely on theinterference of binding of the above-recited motif to DNA but is equallyeffective in the identification of compounds for the above-recitedpurpose. Rather, this embodiment measures directly the interaction of apolypeptide representing GR or of a molecule functionally equivalentthereof with its target DNA which requires a specific binding site forthe GR and is thus, as a rule, different from the DNA that is used as asubstrate for assessing binding by the motif. What is required inaccordance with this assay of the present invention are the compoundsthat are recited under items (aa) to (ac); supra. As regards the(poly)peptide recited in item (aa), the same definition that was used inaccordance with the main embodiment of this invention applies. Inaddition, the (poly)peptide comprises the hsp 70 binding domain of bag-1or a functionally equivalent domain. The hsp70 binding domain of humanbag-1M comprises amino acids 151-264 (Sondermann et al. 2001) of thenaturally occurring molecule. A functionally equivalent domain is alsocapable of binding to hsp 70. For example, such a functionallyequivalent domain may be derived from the naturally occurring domain byan exchange of conservative amino acids. Furthermore, amino acids notcomprised in the binding pocket may be exchanged for different aminoacids with the proviso that that binding is essentially retained (oreven increased). Even amino acids contributing to the binding site forhsp 70 may be exchanged as long as the resulting domain retains thecapacity of binding to hsp 70. An exchange of amino acids may beeffected by use of different methods. For example, site directedmutagenesis may be employed on the DNA basis (see, e.g., Sambrooke etal., “Molecular Cloning, A Laboratory Manual”, CSH Press, Cold SpringHarbor 1989). Once an amino acid exchange has been effected, theresulting molecule would be tested for its binding capacity for hsp 70.Appropriate test systems are available in the art or may be derived fromthis specification. Functionally equivalent domains may also becharacterised by the detection of amino acids and/or the insertion ofadditional amino acids.

With respect to the (poly)peptide mentioned in item (ab) either theglucocorticoid receptor itself or a functionally equivalent molecule maybe employed. Minimal requirements for a functional equivalent of GR arethe DNA binding domain of GR or a functional equivalent thereof, thehinge region of GR or a functional equivalent thereof and atransactivation domain, preferably one or both transactivation domainsof GR (Hollenberg (1988); Mangelsdorf (1995); Savory (2001)). As regardsthe isolation of functionally equivalent molecules, the considerationsmade in connection with the hsp 70 binding domain apply in acorresponding manner here.

Binding sites for the GR have been described in the art, e.g. by Beatoet al. (1989). It is understood that the natural binding site for GR maybe altered in the DNA molecule recited in (ac) as long as its bindingcapacity for the GR is retained. Alterations may be, for example,effected by site-directed mutagenesis (see above) whereupon a bindingassay for GR is carried out. Binding of GR to DNA can be assessed inessence the same way binding of the polypeptide to DNA is assessed, i.e.by using the Biacore technique, electrophoretic mobility shift assays,filter binding assay, or binding of DNA to immobilzed protein or viceversa. Apart from its double-stranded nature, the presence of a GRbinding site is the only necessary requirement for the DNA moleculerecited in (ac). Preferably, said DNA molecule comprises at least 25nucleotides per strand

Conditions that allow complex formation as required by feature (ac) canbe established by the skilled artisan without further ado. Appropriateconditions are, for example, physiological conditions. In addition,various binding conditions have been described in the literature, e.g.Chen et al. (1997), Drouin et al. (1993), Gast et al. (1995), Liu et al.(1995), Ou (2001), Schneikert et al. (1999) or Trapp and Holsboer(1996). Among the parameters varied are the pH (around 7.5),concentrations of NaCl, KCl, EDTA, EGTA, Nonidet P40, dithiothreitol or2-mercaptoethanol, glycerol, poly (dIdC), bovine serum albumine, steroidhormone etc. In addition, different protocols have been used to preparecell extracts containing the glucocorticoid receptor.

In accordance with this embodiment of the present invention, anappropriate compound that inhibits or reduces bag-1 mediateddownregulation of GR may be detected by way of two different read-outsystems represented by options (ba) and (bb). In the first option,binding of the polypeptide representing GR (or of a functionallyequivalent molecule) to the double-stranded DNA is measured (as regardssuitable methodology see above). An increase in binding is indicative ofthe compound having the desired property. According to the secondoption, GR mediated transactivation is preferably measured in reportergene assays in cultivated eukaryotic cells.

In a preferred embodiment of the method of the invention, said(poly)peptides recited in steps (aa) and (ab) and said double-strandedDNA molecule are further contacted with (ad) a (poly)peptide comprisingthe bag-1 binding domain of hsp 70 or a functionally equivalent domainand the GR binding domain of hsp 70 or a functionally equivalent domain;

In this preferred embodiment of the invention, use is made of anadditional compound in the complex formation. The further polypeptidecomprises two domains having a defined specificity. If the naturallyoccurring domains are not employed, then functionally equivalent domainsmay be selected in a corresponding manner as was described for the hsp70 binding domain of bag-1 herein above. If this and the further(poly)peptides described above are non-naturally occurring(poly)peptides, functionality of the domains is nevertheless easilyascertained. For example, functional domains may be linked by a flexiblelinker as is used, for example, in the generation of scFv fragments.Consequently, the complex formation relies on four different compoundsknown to be capable of interaction with one another via specific bindingdomains. By adding hsp70 to the system, compounds can be screened thatdisrupt binding of bag-1 to hsp70.

Another preferred embodiment of the invention relates to a methodwherein the amino acids X and Y comprised in the (poly)peptide recitedin (aa) allow binding of the motif to a double-stranded DNA.

This preferred embodiment envisages that the bag-1 derived motif bindsto DNA. As with the main embodiment of this invention, amino acids (ifany) represented by X and Y support or at least do not prevent saidbinding.

In an additional preferred embodiment of the invention said testcompound or plurality of test compounds is/are further contacted with adouble-stranded DNA molecule that does not comprise a binding site forGR.

This embodiment of the invention is particularly advantageous since itallows not only for assessing modulation of GR transactivation by thecompound to be tested but also for investigating whether said compoundinhibits binding of the motif to DNA. In this way, compounds having thedesired effect which inhibit or do not inhibit binding of the motif maybe differentiated. For example, it is determined whether the effect of acompound that inhibits or reduces bag-1 mediated downregulation of GR isthe result of an inhibition or reduction of the binding of bag-1 to DNAor the result of binding to the recited motif of bag-1 only. In thelatter case, it may be possible that the compound-bag-1 complex stillhas the ability to bind to DNA. However, the binding of the compound tothe recited motif may result, e.g. in a change of the conformation ofbag-1 and, thus, implicate an inhibition or reduction bag-1 mediateddownregulation of GR.

Said double-stranded DNA molecule recited in (ac) further carries areadout-system that is activated upon binding of said GR to said DNA asit is shown in a further preferred embodiment of the invention.

In an additional preferred embodiment of the invention saidreadout-system comprises a reporter gene.

The term “reporter gene” is used in the context of the invention as aterm for a coding unit (gene) whose encoded product (protein) can beeasily assayed (see Glossary of Levin, Genes V, 1994, page 1252). Such areporter gene may be connected to any promoter of interest so thatexpression of the gene can be used to assay the function of the promoterused. In line with the present invention the term “reporter gene” alsocomprises nucleotide sequences coding for fragments of proteins orfusion proteins as long as the promoter dependent expression of saidgenes can be measured in a corresponding assay. Examples for a genewhich can be used as a “reporter gene” are known by a person skilled inthe art and found in the literature, e.g. Mühlhardt, Der Experimentator:Molekularbiologie, Gustav Fischer Verlag 1999.

In a particularly preferred embodiment of the invention said reportergene is selected from a group consisting of firefly luciferase, renillaluciferase, β-galactosidase, human growth hormon (hGH), GFP or anotherfluorescent protein, CAT (chloramphenicolacetyltransferase), alkalinephosphotase including SEAP (secreted alkaline phosphatase), TAT (tyrosylaminotransferase) and peroxidase.

Examples for assays to measure the expression of said reporter genes areknown to a person skilled in the art and described, e.g, in Bronstein etal. (1994), Lewis et al. (1998), Naylor (1999), Schenborn and Groskreutz(1999) or Silverman et al. (1998). Said reporter gene is a gene encodinga transmembrane protein as required in a another particularly preferredembodiment of the invention.

As stated in a most particularly preferred embodiment of the inventionsaid transmembrane protein is a receptor polypeptide.

The expression of said transmembrane protein may be detected by use ofconventional protocols of flow cytometry, histology or other assaysystems are known to a person skilled in the art.

Said test compound(s) is/are selected from the group of small molecules,peptides, aptamers and antibodies or fragments or derivatives thereof inaccordance with a further preferred embodiment of the invention.

The term “aptamers” is well known in the art and described, e.g. inBrody and Gold (2000), Jayasena (1999) or Osborne et al. (1997).Aptamers are preferably single stranded nucleic acid molecules that bindto another molecule as a consequence of their three-dimensionalstructure. Insofar, aptamers have a similar function as antibodies.

“Fragments or derivatives” of antibodies are molecules that retain thebinding specificity of the antibodies. Examples of fragments are Fab orF(ab₂)′ molecules. An example of a derivative is an scFv molecule.

In another particularly preferred embodiment of the invention said smallmolecules are small organic molecules.

Another particularly preferred embodiment shows relates to a methodwherein peptides are derived from an at least partially randomizedpeptide library. Such at least partially randomized peptide librarieshave been described, e.g. in Dolle (2000), Hoppe-Seyler and Butz (2000),Irving et al. (2001) or Kay et al. (2001).

According to a further particularly preferred embodiment of theinvention, said antibodies are monoclonal antibodies.

A further preferred embodiment of the invention relates to a methodwherein, if a plurality of test compounds is tested,

-   (a) different members of said plurality of test compounds are tested    in different reaction vessels wherein those reaction vessels that do    not contain test compounds indicative of being suitable to inhibit    or reduce bag-1 mediated downregulation of GR transactivation are    not further considered;-   (b) members contained in reaction vessels that test positive with    regard to inhibition or reduction of bag-1 mediated downregulation    of GR transactivation are redistributed into different reaction    vessel and tested again; and optionally-   (c) step (b) is repeated until a single compound is identified that    is suitable to inhibit or reduce bag-1 mediated downregulation of GR    transactivation.

In this preferred embodiment of the method of the invention, suitabletest compounds will be identified step by step, if a variety of testcompounds is assayed for the desired activity wherein this variety (i.e.at least two different compounds) are contained in the same lot/reactionvessel. Suitable reaction results include wells of microtiter plateswherein said microtiter plates may have, e.g., 96, 384 or 1536 wells.Reaction vessels not further considered or their contents are usuallydiscarded wherein the required safety standards would be considered.

The assessment referred to above may be effected in an in vitrotranscription/translation system. In this context in vitro systems usingbacteriophage based systems are preferred. More preferably saidbacteriophage based systems using the T7, T3 or Sp6 promoter inaccordance with another preferred embodiment of the invention.

It is known in the art that well established eukaryontic in vitrotranslation systems, e.g. rabbit reticulocyte lysate, wheat germ extractor frog egg extracts, require further adaptations, all within theknowledge of the skilled artesian for the analysis of GR mediatedtransactivation of gene/protein expression. Further systems for analysisof said transactivation, e.g. on the level of transcription, usingeukaryontic promoters will become suitable for the above described invitro systems after further adaptation. For example Ohashi et al. (1994)report of a system using yeast extract, Macias and Stinski (1993)established a system using HeLa cells and Pichon and Christophe (1998)described systems using animal tissue. It is state of the art that saidsystems require extensive synthesis and isolation of differenttranscription factors (Mittler et al. 2001). Thus, further adaption ofsaid systems to the method of the invention is required which is, asstated above, well within the capability of the skilled artesian andrequire no undue burden of work.

In a further preferred embodiment of the method of the presentinvention, the assessment is effected in an eukaryotic cell or tissue oran extract thereof. Suitable eukaryotic cells include immortalized tumorcell lines, listed for example in the catalogues of ATCC and ETCC, inparticular, cell lines that proved to be useful are HeLa, Cos-1, Cos-7,SK-N-MC, and CV-1.

An extract may be obtained from yeast (see, e.g. Ohashi et al. 1994).The approach employing tissue may make use of the system described inPichon and Christophe (1998). Further guidiance in this regard isavailable from Mittler et al. (2001).

The invention relates in another preferred embodiment to a methodwherein X is Met. The recited abbreviations are in accordance with thestandard denomination for amino acids. Thus, Met stands for methionine,Thr for threonine, Ala for alanine.

According to another preferred embodiment, the invention also relates toa method wherein Y is Thr.

In a further preferred embodiment the invention relates to a methodwherein Y is 0.

In another preferred embodiment of the method of the invention, Y isAla-Thr.

Another preferred embodiment of the invention relates to a methodwherein Y is Ala-Ala-Thr.

According to a particularly preferred embodiment of the invention, said(poly)peptide which comprises the motif X-Lys-Lys-Lys-Y-Arg-Arg-Arg isbag-1.

Said (poly)peptide recited in step (ad) is hsp70 in accordance withanother particularly preferred embodiment of the invention.

The present invention also relates in another preferred embodiment to amethod further comprising refining a compound which was identified byany of the above methods of the invention comprises the steps of:

-   (i) identification of the binding site of said compound binding to    said motif and optionally of the binding site of said motif binding    to said compound;-   (ii) molecular modeling of the binding site of the compound and    optionally of the motif; and-   (iii) modification of the compound to improve its binding    specificity for the motif.

All techniques employed in the various steps of the method of theinvention are conventional or can be derived by the person skilled inthe art from conventional techniques without further ado. Thus,biological assays based on the herein identified nature of the compoundsmay be employed to assess the specificity or potency of the (pro)drugs(i.e. compound) wherein the increase of the one or more desiredactivities of the compounds may be used to monitor said specificity orpotency. Steps (1) and (2) can be carried out according to conventionalprotocols. A protocol for site directed mutagenesis is described in LingM M, Robinson B H. (1997) Anal. Biochem. 254: 157-178. The use ofhomology modelling in conjunction with site-directed mutagenesis foranalysis of structure-function relationships is reviewed in Szklarz andHalpert (1997) Life Sci. 61:2507-2520. Chimeric proteins are generatedby ligation of the corresponding DNA fragments, e.g. via a uniquerestriction site using the conventional cloning techniques described inSambrook, Fritsch, Maniatis. Molecular Cloning, a laboratory manual.(1989) Cold Spring Harbor Laboratory Press. A fusion of two DNAfragments that results in a chimeric DNA fragment encoding a chimericprotein can also be generated using the gateway-system (Lifetechnologies), a system that is based on DNA fusion by recombination. Aprominent example of molecular modelling is the structure-based designof compounds binding to HIV reverse transcriptase that is reviewed inMao, Sudbeck, Venkatachalam and Uckun (2000). Biochem. Pharmacol.60:1251-1265.

For example, identification of the binding site of said (pro)drug bysite-directed mutagenesis and chimerical protein studies can be achievedby modifications in the (poly)peptide primary sequence (if the compoundis a (poly)peptide) that affect the drug affinity; this usually allowsto precisely map the binding pocket for the drug.

As regards step (2), the following protocols may be envisaged: Once theeffector site for (pro)drugs has been mapped, the precise residuesinteracting with different parts of the drug can be identified bycombination of the information obtained from mutagenesis studies (step(1)) and computer simulations of the structure of the binding siteprovided that the precise three-dimensional structure of the (pro)drugis known (if not, it can be predicted by computational simulation). Ifsaid (pro)drug is itself a peptide, it can be also mutated to determinewhich residues interact with other residues in the (poly)peptidecomprising the motif.

Finally, in step (3) the (pro)drug can be modified to improve itsbinding affinity or ist potency and specificity. If, for instance, thereare electrostatic interactions between a particular residue of the(poly)peptide comprising the motif and some region of the compound(pro)drug molecule, the overall charge in that region can be modified toincrease that particular interaction.

Identification of binding sites may be assisted by computer programs.Thus, appropriate computer programs can be used for the identificationof interactive sites of a compound and the (poly)peptide comprising themotif by computer assisted searches for complementary structural motifs(Fassina, Immunomethods 5 (1994), 114-120). Further appropriate computersystems for the computer aided design of protein and peptides aredescribed in the prior art, for example, in Berry, Biochem. Soc. Trans.22 (1994), 1033-1036; Wodak, Ann. N.Y. Acad. Sci. 501 (1987), 1-13;Pabo, Biochemistry 25 (1986), 5987-5991. Modifications of the (pro)drugcan be produced, for example, by peptidomimetics and other compoundshaving the same properties can also be identified by the synthesis ofpeptidomimetic combinatorial libraries through successive chemicalmodification and testing the resulting compounds. Methods for thegeneration and use of peptidomimetic combinatorial libraries aredescribed in the prior art, for example in Ostresh, Methods inEnzymology 267 (1996), 220-234 and Dorner, Bioorg. Med. Chem. 4 (1996),709-715. Furthermore, the three-dimensional and/or crystallographicstructure of compounds identified by the method of the invention can beused for the design of peptidomimetic activators, e.g., in combinationwith the (poly)peptide of the invention (Rose, Biochemistry 35 (1996),12933-12944; Rutenber, Bioorg. Med. Chem. 4 (1996), 1545-1558).

Another preferred embodiment of the invention relates to a methodfurther comprising refining a compound as identified or refined hereinabove comprising:

-   (a) modeling said compound by peptidomimetics; and-   (b) chemically synthesizing the modeled compound.

In a further preferred embodiment the invention relates to a methodfurther comprising modifying a compound identified or refined by themethods as described herein above comprising attaching said compound toa signal peptide. Said signal peptide is characterized by its ability toincrease the rate of incorporation of the compound into a cell. Examplesfor said signal peptides are know in the art and described, e.g. inFischer et al. (2000), Lindgren et al. (2000) or Service (2000).

The invention in another preferred embodiment furthermore relates to amethod also comprising modifying a compound identified or refined by themethod as described herein above as a lead compound to achieve (i)modified site of action, spectrum of activity, organ specificity, and/or(ii) improved potency, and/or (iii) decreased toxicity (improvedtherapeutic index), and/or (iv) decreased side effects, and/or (v)modified onset of therapeutic action, duration of effect, and/or (vi)modified pharmakinetic parameters (resorption, distribution, metabolismand excretion), and/or (vii) modified physico-chemical parameters(solubility, hygroscopicity, color, taste, odor, stability, state),and/or (viii) improved general specificity, organ/tissue specificity,and/or (ix) optimized application form and route by (i) esterificationof carboxyl groups, or (ii) esterification of hydroxyl groups withcarbon acids, or (iii) esterification of hydroxyl groups to, e.g.phosphates, pyrophosphates or sulfates or hemi succinates, or (iv)formation of pharmaceutically acceptable salts, or (v) formation ofpharmaceutically acceptable complexes, or (vi) synthesis ofpharmacologically active polymers, or (vii) introduction of hydrophylicmoieties, or (viii) introduction/exchange of substituents on aromates orside chains, change of substituent pattern, or (ix) modification byintroduction of isosteric or bioisosteric moieties, or (x) synthesis ofhomologous compounds, or (xi) introduction of branched side chains, or(xii) conversion of alkyl substituents to cyclic analogues, or (xiii)derivatisation of hydroxyl group to ketales, acetates, or (xiv)N-acetylation to amides, phenylcarbamates, or (xv) synthesis of Mannichbases, imines, or (xvi) transformation of ketones or aldehydes toSchiff's bases, oximes, acetales, ketales, enolesters, oxazolidines,thiozolidines or combinations thereof.

The various steps recited above are generally known in the art. Theyinclude or rely on quantitative structure-action relationship (QSAR)analyses (Kubinyi, “Hausch-Analysis and Related Approaches”, VCH Verlag,Weinheim, 1992), combinatorial biochemistry, classical chemistry andothers (see, for example, Holzgrabe and Bechtold, Deutsche ApothekerZeitung 140 (8), 813-823, 2000).

In another preferred embodiment, the invention relates to a methodfurther comprising producing a pharmaceutical composition comprising thestep of formulating one or more of the compounds identified or refinedby any of the above methods with a pharmaceutically acceptable carrieror diluent.

The pharmaceutical composition produced in accordance with the presentinvention may comprise a pharmaceutically acceptable carrier and/ordiluent. Examples of suitable pharmaceutical carriers are well known inthe art and include phosphate buffered saline solutions, water,emulsions, such as oil/water emulsions, various types of wetting agents,sterile solutions etc. Compositions comprising such carriers can beformulated by well known conventional methods. These pharmaceuticalcompositions can be administered to the subject at a suitable dose.Administration of the suitable compositions may be effected by differentways, e.g., by intravenous, intraperitoneal, subcutaneous,intramuscular, topical, intradermal, intranasal or intrabronchialadministration. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. A typical dose can be, forexample, in the range of 0.001 to 1000 μg (or of nucleic acid forexpression or for inhibition of expression in this range); however,doses below or above this exemplary range are envisioned, especiallyconsidering the aforementioned factors. Generally, the regimen as aregular administration of the pharmaceutical composition should be inthe range of 1 μg to 10 mg units per day. If the regimen is a continuousinfusion, it should also be in the range of 1 μg to 10 mg units perkilogram of body weight per minute, respectively. Progress can bemonitored by periodic assessment. Dosages will vary but a preferreddosage for intravenous administration of DNA is from approximately 10⁶to 10¹² copies of the DNA molecule. The compositions of the inventionmay be administered locally or systemically. Administration willgenerally be parenterally, e.g., intravenously; DNA may also beadministered directly to the target site, e.g., by biolistic delivery toan internal or external target site or by catheter to a site in anartery. Preparations for parenteral administration include sterileaqueous or non-aqueous solutions, suspensions, and emulsions. Examplesof non-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition producedin accordance with the invention may comprise further agents such asinterleukins or interferons depending on the intended use of thepharmaceutical composition.

The invention finally relates to the use of a compound identified by themethod of the invention or refined by any of the methods recited abovefor the preparation of a pharmaceutical composition for the preventionor treatment of Cushing syndrome, multiple sclerosis, asthma, arthritis,other inflammatory diseases or depression.

Said pharmaceutical composition may, optionally, comprise one or morepharmaceutically acceptable carrier or diluents, e.g., as have beendescribed herein above.

Examples of other inflammatory diseases are sterile or infectiousinflammations such as poststreptococcal autoimmune inflammation(autoimmune nephritis etc.), sepsis, viral and bacterial infections,fungi, parasites, graft rejection. TABLE 1 Structure of BAG-1M, BAG-1Sand the mutants BAG-1MΔN10 and BAG-1MD11-67. DNA = DNA-binding domain,UBI: Ubiquitin- like-domain, hsp70-IAD: Hsp70-Interacting-domain, E₂X₄:E₂X₄-domains.

Table 1:Structure of BAG-1M, BAG-1S and the mutants BAG-1MΔN10 and BAG-1MΔ11-67.DNA = DNA-binding domain,UBI: Ubiquitin-like-domain,hsp70-IAD: Hsp70-Interacting-domain,E₂X₄: E₂X₄-domains:

TABLE 2 Structure of BAG-1 mutated in the DNA binding domain.

Table 2 Structure of BAG-1M, mutant BAG-1MΔ11-67, and the mutants of theDNA-binding domain of BAG-1, BAG-1MKA, BAG-1M-T5, BAG-1M+A5, BAG-1M+2A5UBI: Ubiquitin-like-domain,hsp70-IAD: Hsp7O-Interacting-domain,E₂X₄: E₂X₄-domains.

The figures show:

FIG. 1:

Effect of wt BAG-1M, ΔN10 and Δ11-67 on GR-dependent transcription. ASK-N-MC cells were transiently transfected with 3.5 μg of GR-responsiveMMTV-luciferase (MTV-Luc) indicator gene, 1 μg of an internal controlplasmid encoding renilla luciferase under the control of theSV40-promoter and 4 μg of a human GR-encoding plasmid (pRK7GRHA). Inaddition, 6 μg of either an empty expression vector (C=control) or themutants BAG-1MΔN10, BAG-1MΔ11-67 or the isoforms BAG-1M and BAG-1S werecotransfected with a total amount of 14.5 μg transfected plasmid DNA ineach sample. Cells were transfected using electroporation, replated andcultured for 24 h in fresh medium either with or without 100 nMdexamethasone. Luciferase activities were corrected byRenilla-luciferase activities and are presented as percent activity withthe activity of control vector-transfected cells set as 100%. Resultsrepresent mean values±s.e.m. of eight independent experiments performedin sextuplicate. B/C Representative western blot of either GR- orBAG-protein. Whole cell extracts used for luciferase assays wereprepared for SDS-PAGE and immunoblotting. Antibodies were directedagainst either the C-terminus of BAG-1 or the HA-tag on GR.

FIG. 2:

A/B Bacterially expressed BAG-1 and mutants. BAG-1 isoforms and mutantswere cloned into the bacterial expression vector pProexHTa (Lifetech.Inc., Rockeville, USA) that delivered a histidine-tail to the N-terminusof the proteins. BAG-1M isoforms and mutants were expressed in E. coli,purified using Ni-Agarose columns following the QIAexpressionistprotocol (QIAGEN Inc., Hilden, Germany). The histidine-tail was removedby cleaving with tobacco edge virus protease and proteins were analysedby SDS-PAGE 12% and 15%. Staining was performed with Coomassie blueR250. Precleaved and cleaved proteins were applied alternately ontogels. A: BAG-1M=M, BAG-1MKA=KA, BAG-1M-T5=−T5, BAG-1M+A5=+A5,BAG-1M+2A5=+2A5 B: BAG-1MΔ9-67, BAG-1MΔ11-67=Δ11-67, BAG-1S=S,BAG-1MΔN10=ΔN10. C/D DNA-binding of BAG-1M isoforms and mutants. Aradiolabeled 125 bp fragment from phage λ-DNA/HINDIII was eitheremployed as such (lane 0) or used for electrophoretic mobility-shiftassays with the cleaved proteins, either 1 μg (a-lanes) or 2 μg(b-lanes) were allowed to bind to ³²P-labelled DNA for 30 min. at 25° C.and complexes were separated on an acrylamide gel under nativeconditions. Shown are representative autoradiograms. C: Full lengthBAG-1M=M, BAG-1MKA=KA, BAG-1M=ΔN10, BAG-1MΔ9-67=Δ9-67 and D:BAG-1MΔ11-67=Δ11-67, BAG-1S=S, BAG-1M-T5=−T5, BAG-1M+A5=+A5,BAG-1M+2A5=+2A5.

FIG. 3:

DNA binding-defective mutants are unable to inhibit GR. A SK-N-MC cellswere transiently transfected with 3.5 μg of GR-responsiveMMTV-luciferase (MTV-Luc) indicator gene, 1 μg of an internal controlencoding renilla luciferase under the control of the SV40-promoter and 4μg of a human GR-encoding plasmid (pRK7GRHA). In addition, 6 μg ofeither an empty expression vector (C=control) or the mutants BAG-1MKA,BAG-1MΔ9-67 BAG-1M+A5 BAG-1M+2A5 BAG-1M-T5 resp. the wildtype BAG-1Mwere cotransfected, with the total amount of 14.5 μg transfected plasmidDNA in each sample. Cells were transfected by electroporation, replatedand cultured for 24 h in fresh medium either with or without 100 nMdexamethasone. Luciferase activities were corrected byRenilla-luciferase activities and are presented as percent activity withthe firefly-luciferase activity of control vector-transfected cells setas 100%. Results represent mean values±s.e.m. of six independentexperiments performed in sextuplicate. B/C Representative western blotof either GR- or BAG-protein. Whole cell extracts used for luciferaseassays were prepared for SDS-PAGE and immunoblotting. Antibodies weredirected against either the C-terminus of BAG or the HA-tag on GR.

FIG. 4:

The DNA binding domain and the hsp70 interaction domain of BAG-1 need tobe present in cis. A SK-N-MC cells were transiently transfected with 3.5μg of GR-responsive MMTV-firefly luciferase (MTV-Luc) indicator gene,2.5 μg of an internal control plasmid encoding β-galactosidase(pCMVβGal) under the control of the CMV-promoter and 4 μg of a humanGR-encoding plasmid (pRK7GRHA). In addition, 6 μg of either an emptyexpression vector (C=control) or the mutants BAG-1Mhsp70mut (here:70mut), BAG-1MKA or the wildtype BAG-1M were cotransfected, either 6 μgof the respective plasmid alone (lane2-4), of each 3 μg resp. 6 μgBAG-1MKA and BAG-1Mhsp70mut together (lane 5 resp. 7) or 3 μg resp. 6 μgof BAG-1Mhsp70mut and BAG-1M together (lane 6 resp. 8). Cells weretransfected using electroporation, replated and cultured for 24 h infresh medium either with or without 100 nM dexamethasone. Luciferaseactivities were corrected by β-galactosidase activities and arepresented as percent activity with the firefly-luciferase activity ofcontrol vector-transfected cells set as 100%. Results represent meanvalues±s.e.m. of four independent experiments performed in sextuplicateB/C Representative western blot of either GR- or BAG-protein. Whole cellextracts used for luciferase assays were prepared for SDS-PAGE andimmunoblotting. Antibodies were directed against either the C-terminusof BAG or the HA-tag on GR.

FIG. 5:

Model for the inhibitory mechanism of BAG-1M on GR employing its DNAbinding and hsp70 interaction domains. Upon activation of the receptorby hormone, BAG-1M and hsp70 are translocated into the nucleus. GRinteracts with its binding sites and associated factors open thechromatin structure. This allows BAG-1M to bind to DNA, therebyinhibiting the further functions of GR in the process of transcriptionalactivation.

The examples illustrate the invention.

FIG. 6:

Amino acid sequence of human BAG1; Genbank Accession No. Q99933

FIG. 7:

Amino acid sequence of human GRα; Genbank Accession No.: AAB64353

FIG. 8:

Amino acid sequence of human GRβ; Genbank Accession No.: AAB64354

EXAMPLE 1 Deletion of the N-Terminal DNA Binding Domain of BAG-1Abolishes its Inhibitory Function on GR, While Deletion of the E₂X₄Motif Domain Does Not

BAG-1S, the short isoform of BAG-1, is unable to inhibit thetranscriptional activity of GR (Schneikert et al., 1999). The N-terminalamino acid stretch missing in BAG-1S as compared to BAG-1M contains aserine- and threonine-rich E₂ X₄ repeat domain and a recently describedDNA binding domain (Table 1 and (Zeiner et al., 1999)). To begin tounderstand which features in the N-terminus are required for inhibitionof GR, two deletion mutants were created, either missing the E₂X₄ domain(BAG-1M Δ11-67, Table 1) or the putative DNA binding domain (BAG-1MΔN10, FIG. 1). To this effect, a series of BAG-1 mutants was generatedby PCR and cloned into the mammalian expression plasmid pRK5mcs; most ofthe mutants were cloned also into pcDNA4TO (InVitrogen), which allowsinducible expression in cells containing the Tet-repressor. The plasmidpRK5mcs was derived from pRK5SV40PUR (Spengler et al., 1993) by cuttingwith the restriction endonucleases EcoRI and HindIII and inserting anannealed linker oligonucleotide with the sequences5′-AATTCTCGAGATATCGGGCCCGGATCCGCGGCCGCTCGCGA-3′ and5′AGCTTCGCGAGCGGCCGCGGATCCGGGCCCGATATCTCGAG-3′ for the opposite strand.The wild-type and mutant sequences of BAG-1 were amplified by PCR fromthe clone (Höhfeld and Jentsch, 1997) using the oligonucleotide 5′ TCCTCT AGA TCA CTC GGC CAG GGC AAA GTT TG 3′ (for cloning into pcDNA4TO) or5′ TCC GGA TCC CTC GGC CAG GGC AAA GTT TG 3′ (for cloning into pRK5mcs)as downstream primers and one of the following oligonucleotides asupstream primers: BAG-1 form Oligonucleotide sequence 5′ to 3′ wt BAG-1MTCG GAA TTC ATG AAG AAG AAA ACC CG Δ N10 CCA GAA TTC ATG CGG AGC GAG GAGTTG A Δ 11-67 TCC GAA TTC ATG AAA AAG AAG ACT CGC CGG CGG AGT ACA ATGGCG GCA GCT GGG CTC Wt BAG-1S TCC GAA TTC ATG GCG GCA GCT GGG CTC Δ 9-67TCC GAA TTC ATG AAA AAG AAG ACT CGC CGG CGG ATG GCG GCA GCT GGG CTC KATCC GAA TTC ATG GCT GCC GCA ACC CGG CGC TCG ACC Δ T₅ TCG GAA TTC ATG AAGAAG AAA CGG CGC CGC TCG ACC C +A₅ TCG GAA TTC ATG AAG AAG AAA GCT AGCCGG CGC CGC TCG A +2A₅ TCG GAA TTC ATG AAG AAG AAA GCT GCA ACC CGG CGCCGC TCG A

The PCR products were cut with the restriction endouncleases EcoRI andBamHI for cloning into pRK5mcs and cut with EcoRI and XbaI for cloninginto pcDNA4TO and gel-purified (Qiaquick, Gel-extraction kit, QiagenCorp.). Most expression plasmids were created with and without a FLAGtag. The FLAG-tag was put to the N-terminus, because a C-terminal FLAGturned out to be unstable. For all FLAG constructs, the sameoligonucleotides were used as listed above except that the sequence infront of the first ATG was replaced by the sequence 5′ TCC GAA TTC ATGGAC TAC AAG GAC GAC GAT GAC AAG 3′. For expression in bacteria thevector pProExHTa (Life Technologies) was chosen. The downstream primercontained the sequence: 5′ TCT GTA TCA GGC TGA AAA TCT TCT CTC 3′, theNcoI and the XbaI site was used for cloning and, therefore, the upstreamprimers for PCR amplification contained the sequences depicted in thefollowing ′ in front of the first ATG.

These mutants were analysed in transient transfection assays in two celllines, COS-7 cells and the neuroblastoma cell line SK-N-MC. Cell cultureand transfiction were carried out as follows: Human neuroblastomaSK-N-MC cells (ATCC # HTB-10) and COS-7 cells lines were cultured inDMEM supplemented with 10% fetal calf serum (FCS), 36 mg/l sodiumpyruvate, 100 units/ml penicillin, 100 μg/ml streptomycin sulfate, 0.25μg/ml amphothericin (all from Life Technologies, Inc.) and 4.5 g/lglucose at 37° C. and 10% CO₂.

Two days before transfection, cells were seeded into medium containing10% charcoal-stripped, steroid-free FCS. Dextran T-70 (Pharmacia,Uppsala, Sweden) was used for charcoal-stripping of FCS (Damm, 1994).

Cells were harvested at about 70-90% confluency and about 0.5 to 1×10⁷cells were resuspended in 400 μl of electroporation buffer (50 mMK₂HPO₄, 20 mM KAc, pH 7.35). 3.5 μg steroid-responsive fireflyluciferase reporter plasmid MTV-Luc (Hollenberg and Evans, 1988), 4 μgpRK7GR that expresses human GR (Hollenberg et al., 1985) from theCMV-promoter of the vector pRK7 (Spengler et al., 1993), 6 μg of eitherone of the BAG-1 expression plasmids (Tables 1 and 2) or thecorresponding empty expression vector and, as internal control plasmids,either 3.5 μg simian virus 40 (SV40) promoter-driven β-galactosidaseexpression vector pCH110 (Pharmacia LKB, Freiburg, Germany) or 1 μg ofSV40-driven renilla luciferase driven expression vector (Promega) wereadded and transfection was performed using an electroporation system(Biotechnologies & Experimental Research, San Diego, Calif.)—afterdetermination of the optimal electrical field strength (Chu et al.,1987). Electroporated cells were replated and cultured for 24 hours infresh medium (containing 10% steroid-free FCS), supplemented with 100 nMdexamethasone (Sigma-Aldrich) or the solvent of dexamethasone (i.e.ethanol).

Cells were expressed with a reporter plasmid carrying the luciferasegene driven by the GR-sensitive mouse mammary tumor virus (MMTV)promoter, a renilla luciferase reference plasmid and either an emptyexpression vector or a vector expressing one of the BAG-1M mutants. Thefirefly luciferase and β-galactosidase assays were as described before(Herr et al., 2000). Either cells were scraped in 200 μl of lysis buffer(0.1 M KHPO₄, pH 7.8, 1 mM DTT) and cytosolic extracts were made bythree freeze and thaw cycles and subsequent centrifugation. 50 μl ofeach supernatant (corresponding to ˜1-2×10⁵ cells) were transferred to a96 well plate. 150 μl of 33 mM KHPO₄, pH 7.8, 1.7 mM ATP, 3.3 mM MgCl₂,13 mM Luciferin (Roche Biochemicals, Mannheim, Germany) was added toeach sample by the injector of an automatic luminometer (Luminat LB 96,Wallac GmbH, Freiburg, Germany) and light emission was measured for 10seconds. To correct for variations in transfection efficiencies, valuesof the luciferase assay were normalized using β-galactosidase activitiesthat were measured as follows: 50 μl of cell extract was added to 100 μlgalactosidase buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1 mMMgCl₂, 50 mM β-mercaptoethanol) on a 96 well plate. 20 μl of 2 mg/mlONPG was added and the reaction was incubated at 37° C. After 10-30minutes, absorption was measured at 405 nm in a multiphotometer(Dynatech MR5000).

Or, when using the renilla luciferase, expression plasmid in combinationwith the firefly reporter plasmid, cell extracts were scraped in 200 μlof a lysis buffer provided and prepared according to the manufacturersrecommendation (Promega Inc.) and firefly and renilla luciferaseactivities were measured on a 96-well plate in 100 μl cell extract inthe automatic luminometer.

BAG-1M clearly inhibited GR-dependent reporter gene transcription, asexpected (FIG. 1A). Surprisingly, however, deletion of the E₂X₄ 1A) hadno influence on the ability of bag-1 to inhibit the transcriptionalactivity of GR (FIG. 2A). On the other hand, deletion of the N-terminal10 amino acids completely abolished the effect of BAG-1 on GR. BAGproteins without the N-terminal FLAG peptides gave the same results.Also, the same pattern of activity of the BAG-1 mutants was observed inCOS-7 cells and HeLa-cells. Since co-expression of various plasmids canproduce misleading results in case of expression interference (Hofman etal., 2000), the relative amounts of GR and of BAG-1 were rechecked. Theprotein levels of GR were similar in all the experiments (FIG. 1B).Similarly, the levels of the expressed BAG-1 proteins were comparablethroughout our experiments (FIG. 1C). Therefore, the domain recently putforward as DNA binding domain of BAG-1 (Zeiner et al., 1999) isnecessary for the inhibitory effect of BAG-1 on GR.

EXAMPLE 2 Spacing of the Lysine and Arginine Stretch is Not Importantfor DNA Binding of BAG-1

To characterise the DNA binding domain of BAG-1 in more detail, a seriesof additional mutants were created (FIG. 3); all proteins (FIGS. 1 and3) derived from BAG-1 mutants and isoforms in table 1 and 2 wereexpressed in bacteria and purified (FIG. 2A/B). As a reference, lysines2, 3 and 4 were mutated to alanines (BAG-1M KA), which has beendescribed to abolish DNA binding of BAG-1. To confine the DNA bindingdomain essentially to the positively charged amino acids lysine andarginine, amino acids 9 to 67 (Δ9-67) were deleted. Moreover, thespacing between lysines 2, 3, 4 and arginines 6, 7, 8 was changed byeither deleting threonine 5 (BAG-1M −T₅) or inserting one (BAG-1M +A₅)or two alanines-(BAG-1M +2A₅). After expression in bacteria, proteinswere purified using Ni-NTA agarose (FIG. 2A/B;a-lanes) and the histidinetails were cleaved with tobacco etch virus protease (FIG. 2A/B;b-lanes).To this effect, Histidine-tagged BAG-1 encoding plasmids were grown inpBL3Lys-bacteria (Life Technologies). Lysis and purification wasperformed using Ni-NTA agarose according to the manufacturer'srecommendations (Qiagen). The histidine tag was removed by cleavage withTEV protease for 6 h at 30° C. (Life Technologies). Then, cleavedproteins were rebound to Ni-NTA agarose columns again to remove tracesof uncleaved proteins and purified afterwards with Biospin columns(Bio-Rad) to remove DDT from cleavage-buffer and traces of liquidcolumns. Protein concentrations were determined using theBCA-Protein-Assay-Kit (Pierce).

The purified and cleaved proteins were used to examine their ability tobind to DNA. The 125 base pair fragment of a Hind III cleavage of DNAfrom bacteriophage Δ was chosen as template. Gel shift assays (FIG. 4C)revealed that mutation of lysines 2, 3 and 4 to alanines abolished DNAbinding, consistent with a recent report (Zeiner et al., 1999).Experimentally, DNA binding of BAG-1 was essentially performed asdescribed (Zeiner, Niyaz, et al. 1999). Briefly, 1 μg or 2 μg ofpurified and TEV-cleaved BAG-protein (isoforms or mutant) were incubatedwith 0.2 ng of 32P-end-labeled 125 bp λ/HindIII DNA fragment in bindingbuffer (Zeiner, Niyaz, et al. 1999) for 30 min. at RT. Protein DNAcomplexes were resolved on native 5% acrylamide gels in TBE buffer.

However, deletion of the E₂X₄ domain (BAG-1M Δ11-67) had no influence onDNA binding. Even additional deletion of two N-terminal amino acidsretained DNA binding (BAG-1M Δ9-67). BAG-1S, as expected, did not bindto DNA. Therefore, it appears that the first 8 N-terminal amino acidsare sufficient to confer the ability of BAG-1 to bind to DNA. Moreover,spacing between the positively charged amino acids lysines 2, 3, 4 andarginines 6, 7, 8 is not important for DNA binding, because thesemutants bind to DNA as efficiently as BAG-1M (FIG. 2C/D). Therefore,BAG-1M apparently contains a short, unusual DNA binding domain.

EXAMPLE 3 Mutants that Inhibit DNA Binding of BAG-1 also Abolish itsInhibitory Function on GR

The results shown in FIG. 1 suggest that DNA binding of BAG-1 isnecessary for its inhibitory effect on GR function. We set out to eitherstrengthen this correlation or prove that, while this domain per se isnecessary, it is not DNA binding, but some other property of this domainthat causes the inhibitory effect. Therefore, expression clones of allmutants, with and without FLAG-tag were constructed, and tested in thetransient reporter gene assay as described in FIG. 2. BAG-1M Δ9-67,BAG-1M −T₅, BAG-1M +A₅ and BAG-1M +2A₅ all were able to inhibitGR-dependent transcription (FIG. 3A). In contrast, BAG-1S and BAG-1M KAlost the inhibitory effect on GR. Again, the protein levels of GR werecomparable throughout the experiments (FIG. 3B), as were the levels ofthe different BAG-1 mutants (FIG. 3C). The results with BAG-1 mutantswithout a FLAG tag showed the same pattern. Also, similar data wereobtained in HeLa cells.

Therefore, the data in FIGS. 1, 2 and 3 clearly demonstrate that allmutants of BAG-1M that are able to bind to DNA inhibit thetranscriptional activity of GR, while those that are unable to bind toDNA have no influence on GR activity.

EXAMPLE 4 The DNA Binding and the hsp70 Interaction Domain of BAG-1 Needto be Present in cis to Inhibit GR Function

Besides the N-terminal domain, deletion of the C-terminal 70 amino acidsalso abolishes the effect of BAG-1 on GR (Schneikert et al., 2000).Since these amino acids contain the interaction domain with hsp70, ithas been proposed that interaction with hsp70 is necessary for thefunction of BAG-1. This raises the question whether the domainsresponsible for interaction with hsp70 and for binding to DNA arerequired to be present in cis. Therefore, a point mutation of BAG-1 thatabolishes interaction with hsp70 (R237A BAG-1M (Sondermann et al., 2001)and data not shown) was first created.

In the above-recited transient reporter gene assay, this mutantabolishes the inhibition of GR activity by BAG-1 (FIG. 4A, lane 4). Thisproves that the inability to interact with hsp70 rather than some other,concomitant consequence is the correct explanation for the effect ofC-terminal deletions of BAG-1. To test whether a BAG-1 protein that isunable to interact with hsp70, but has an intact DNA binding domain, canrescue the inhibitory function of a DNA binding mutant bearing an intacthsp70 interaction domain, a DNA binding mutant was concomitantlyexpressed with an hsp70 interaction mutant in the reporter assay.Although these proteins are expressed to the same or even higher levelcompared to wt BAG-1 (FIG. 4B), no inhibition of GR-dependent reportergene transcription was detected (FIG. 4A, lanes 5+6). The levels of GRwere the same throughout the experimental conditions (FIG. 4C). It isconcluded that the N-terminal domain and the hsp70 interaction domain ofBAG-1 need to be present at the same time and on the same molecule.

REFERENCE LIST

-   Ballinger, C. A., Connell, P., Wu, Y., Hu, Z., Thompson, L. J.,    Yin, L. Y., and Patterson, C. (1999). Identification of CHIP, a    novel tetratricopeptide repeat-containing protein that interacts    with heat shock proteins and negatively regulates chaperone    functions. Mol Cell Biol 19, 4535-4545.-   Beato, M.; Chalepakis, G.; Schauer, M.; Slater, E. P. DNA regulatory    elements for steroid hormones. (1989). J. Steroid Biochem. 32,    737-747-   Bimston, D., Song, J., Winchester, D., Takayama, S., Reed, J. C.,    and Morimoto, R. I. (1998). BAG-1, a negative regulator of Hsp70    chaperone activity, uncouples nucleotide hydrolysis from substrate    release. EMBO J. 17, 6871-6878.-   Bresnick, E. H., Dalman, F. C., Sanchez, E. R., and Pratt, W. B.    (1989). Evidence that the 90-kDa heat shock protein is necessary for    the steroid binding conformation of the L cell glucocorticoid    receptor. J. Biol. Chem. 264, 4992-4997.-   Briknarova, K., Takayama, S., Brive, L., Havert, M. L., Knee, D. A.,    Velasco, J., Homma, S., Cabezas, E., Stuart, J., Hoyt, D. W.,    Satterthwait, A. C., Llinas, M., Reed, J. C., and Ely, K. R. (2001).    Structural analysis of BAG1 cochaperone and its interactions with    Hsc70 heat shock protein. Nat. Struct. Biol. 8, 349-352.-   Brody, E N and Gold, L (2000) Aptamers as therapeutic and diagnostic    agents. J Biotechnol 74, 5-13.-   Bronstein, I, Fortin, J, Stanley, P E, Stewart, G S, Kricka, L    J (1994) Chemiluminescent and bioluminescent reporter gene assays.    Anal Biochem 219, 169-181.-   Buchner, J. (1999). Hsp90 & Co.—a holding for folding. Trends    Biochem Sci 24, 136-141.-   Chen, S, Wang, J, Yu, G, Liu, W, Pearce, D (1997) Androgen and    glucocorticoid receptor heterodimer formation. A possible mechanism    for mutual inhibition of transcriptional activity. J Biol Chem 272,    14087-14092.-   Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the    efficient transfection of mammalian cells with DNA. Nucleic Acids    Res 15, 1311-1326.-   Damm, K. (1994). Gene transfection studies using recombinant steroid    receptors. In Methods in Neurosciences: Neurobiology of    Steroids. E. R. De Kloet and W. Sutanto, eds. (San Diego: Academic    Press), pp. 265-276.-   Dittmar, K. D., Banach, M., Galigniana, M. D., and Pratt, W. B.    (1998). The role of DnaJ-like proteins in glucocorticoid    receptor.hsp90 heterocomplex assembly by the reconstituted    hsp90.p60.hsp70 foldosome complex. J. Biol. Chem. 273, 7358-7366.-   Dittmar, K. D., Demady, D. R., Stancato, L. F., Krishna, P., and    Pratt, W. B. (1997). Folding of the glucocorticoid receptor by the    heat shock protein (hsp) 90-based chaperone machinery. The role of    p23 is to stabilize receptor.hsp90 heterocomplexes formed by    hsp90.p60.hsp70. J. Biol. Chem. 272, 21213-21220.-   Dittmar, K. D., Hutchison, K. A., Owens-Grillo, J. K., and    Pratt, W. B. (1996). Reconstitution of the steroid receptor.hsp90    heterocomplex assembly system of rabbit reticulocyte lysate. J.    Biol. Chem. 271, 12833-12839.-   Dolle, R E (2000) Comprehensive survey of combinatorial library    synthesis: 1999. J Comb Chem 2, 383-433.-   Drouin, J, Sun, Y L, Chamberland, M, Gauthier, Y, De Lean, A, Nemer,    M, Schmidt, T J (1993) Novel glucocorticoid receptor complex with    DNA element of the hormone-repressed POMC gene. EMBO J. 12, 145-156.-   Fischer, P. M., Zhelev, N. Z., Wang, S., Melville, J. E., Fahraeus,    R., and Lane, D. P. (2000). Structure-activity relationship of    truncated and substituted analogues of the intracellular delivery    vector Penetratin. J. Pept. Res. 55,163-172.-   Freedman, L. P. (1999). Increasing the complexity of coactivation in    nuclear receptor signaling. Cell 97, 5-8.-   Freeman, B. C., Felts, S. J., Toft, D. O., and Yamamoto, K. R.    (2000). The p23 molecular chaperones act at a late step in    intracellular receptor action to differentially affect ligand    efficacies. Genes Dev 14, 422-434.-   Freeman, B. C., Myers, M. P., Schumacher, R., and Morimoto, R. I.    (1995). Identification of a regulatory motif in Hsp70 that affects    ATPase activity, substrate binding and interaction with HDJ-1.    EMBO J. 14, 2281-2292.-   Froesch, B. A., Takayama, S., and Reed, J. C. (1998). BAG-1L protein    enhances androgen receptor function. J Biol Chem 273, 11660-11666.-   Gast, A, Neuschmid-Kaspar, F, Klocker, H, Cato, A C (1995) A single    amino acid exchange abolishes dimerization of the androgen receptor    and causes Reifenstein syndrome. Mol Cell Endocrinol 111, 93-98.-   Gebauer, M., Zeiner, M., and Gehring, U. (1998). Interference    between proteins Hap46 and Hop/p60, which bind to different domains    of the molecular chaperone hsp70/hsc70. Mol. Cell Biol. 18,    6238-6244.-   Guzey, M., Takayama, S., and Reed, J. C. (2000). BAG1L enhances    trans-activation function of the vitamin D receptor. J. Biol. Chem.-   Herr, A., Wochnik, G. M., Rosenhagen, M. C., Holsboer, F., and    Rein, T. (2000). Rifampicin is not an activator of the    glucocorticoid receptor. Mol. Pharmacol. 57, 732-737.-   Hofman, K., Swinnen, J. V., Claessens, F., Verhoeven, G., and    Heyns, W. (2000). Apparent coactivation due to interference of    expression constructs with nuclear receptor expression. Mol. Cell    Endocrinol. 168, 21-29.-   Hollenberg, S. M. and Evans, R. M. (1988). Multiple and cooperative    trans-activation domains of the human glucocorticoid receptor. Cell    55, 899-906.-   Hollenberg, S. M., Weinberger, C., Ong, E. S., Cerelli, G., Oro, A.,    Lebo, R., Thompson, E. B., Rosenfeld, M. G., and Evans, R. M.    (1985). Primary structure and expression of a functional human    glucocorticoid receptor cDNA. Nature 318, 635-641.-   Höhfeld, J. and Jentsch, S. (1997). GrpE-like regulation of the    hsc70 chaperone by the anti-apoptotic protein BAG-1 [published    erratum appears in EMBO J. 1998 Feb. 2; 17 (3):847]. EMBO J. 16,    6209-6216.-   Höhfeld, J., Minami, Y., and Hartl, F. U. (1995). Hip, a novel    cochaperone involved in the eukaryotic Hsc70/Hsp40 reaction cycle.    Cell 83, 589-598.-   Hoppe-Seyler, F Butz, K (2000) Peptide aptamers: powerful new tools    for molecular medicine. J Mol Med 78, 426-430.-   Irving, M B, Pan, O, Scott, J K (2001) Random-peptide libraries and    antigen-fragment libraries for epitope mapping and the development    of vaccines and diagnostics. Curr Opin Chem Biol 5, 314-324.-   Jayasena, S D (1999) Aptamers: an emerging class of molecules that    rival antibodies in diagnostics. Clin Chem 45, 1628-1650.-   Kanelakis, K. C., Murphy, P. J., Galigniana, M. D., Morishima, Y.,    Takayama, S., Reed, J. C., Toft, D. O., and Pratt, W. B. (2000).    hsp70 interacting protein hip does not affect glucocorticoid    receptor folding by the hsp90-based chaperone machinery except To    oppose the effect of BAG-1 [In Process Citation]. Biochemistry 39,    14314-14321.-   Kay, B K, Kasanov, J, Yamabhai, M (2001) Screening phage-displayed    combinatorial peptide libraries. Methods 24, 240-246.-   Knee, D. A., Froesch, B. A., Nuber, U., Takayama, S., and    Reed, J. C. (2001). Structure-function analysis of bag1 proteins.    effects on androgen receptor transcriptional activity. J. Biol.    Chem. 276, 12718-12724.-   Kullmann, M., Schneikert, J., Moll, J., Heck, S., Zeiner, M.,    Gehring, U., and Cato, A. C. (1998). RAP46 is a negative regulator    of glucocorticoid receptor action and hormone-induced apoptosis. J.    Biol. Chem. 273, 14620-14625.-   Lewis, J C, Feltus, A, Ensor, C M, Ramanathan, S, Daunert, S (1998)    Applications of reporter genes. Anal Chem 70, 579A-585A.-   Lindgren, M., Hallbrink, M., Prochiantz, A., and Langel, U. (2000).    Cell-penetrating peptides. Trends. Pharmacol. Sci. 21, 99-103.-   Liu, J. and DeFranco, D. B. (1999). Chromatin recycling of    glucocorticoid receptors: implications for multiple roles of heat    shock protein 90. Mol. Endocrinol 13, 355-365.-   Liu, R., Takayama, S., Zheng, Y., Froesch, B., Chen, G. Q., Zhang,    X., Reed, J. C., and Zhang, X. K. (1998). Interaction of BAG-1 with    retinoic acid receptor and its inhibition of retinoic acid-induced    apoptosis in cancer cells. J. Biol. Chem. 273, 16985-16992.-   Liu, W, Wang, J, Sauter, N K, Pearce, D 1995 Steroid receptor    heterodimerization demonstrated in vitro and in vivo. Proc Natl Acad    Sci USA 92, 12480-12484.-   Mangelsdorf, D J, Thummel, C, Beato, M, Herrlich, P, Schutz, G,    Umesono, K, Blumberg, B, Kastner, P, Mark, M, Chambon, P (1995). The    nuclear receptor superfamily: the second decade. Cell 83, 835-839.-   McNally, J. G., Muller, W. G., Walker, D., Wolford, R., and    Hager, G. L. (2000). The glucocorticoid receptor: rapid exchange    with regulatory sites in living cells. Science 287, 1262-1265.-   Michels, A. A., Kanon, B., Bensaude, O., and Kampinga, H. H. (1999).    Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian    cells. J. Biol. Chem. 274, 36757-36763.-   Michels, A. A., Kanon, B., Konings, A. W., Ohtsuka, K., Bensaude,    O., and Kampinga, H. H. (1997). Hsp70 and Hsp40 chaperone activities    in the cytoplasm and the nucleus of mammalian cells. J. Biol. Chem.    272, 33283-33289.-   Mittler, G, Kremmer, E, Timmers, H T, Meisterernst, M (2001) Novel    critical role of a human Mediator complex for basal RNA polymerase    11 transcription. EMBO Rep 2, 808-813.-   Naylor, L H 1999 Reporter gene technology: the future looks bright.    Biochem Pharmacol 58, 749-757.-   Niyaz, Y., Zeiner, M., and Gehring, U. (2001). Transcriptional    activation by the human Hsp70-associating protein Hap50. J. Cell    Sci. 114, 1839-1845.-   Nollen, E. A., Brunsting, J. F., Song, J., Kampinga, H. H., and    Morimoto, R. I. (2000a). Bag1 functions In vivo as a negative    regulator of hsp70 chaperone activity. Mol Cell Biol 20, 1083-1088.-   Nollen, E. A., Kabakov, A. E., Brunsting, J. F., Kanon, B., Hohfeld,    J., and Kampinga, H. H. (2000b). Modulation of in vivo HSP70    chaperone activity by Hip and BAG-1. J. Biol; Chem.-   Ohashi, Y, Brickman, J M, Furman, E, Middleton, B, Carey, M (1994)    Modulating the potency of an activator in a yeast in vitro    transcription system. Mol Cell Biol 14, 2731-2739.-   Osborne, S E, Matsumura, I, Ellington, A D (1997) Aptamers as    therapeutic and diagnostic reagents: problems and prospects. Curr    Opin Chem Biol 1, 5-9.-   Ou, X M, Storring, J M, Kushwaha, N, Albert, P R (2001).    Heterodimerization of mineralocorticoid and glucocorticoid receptors    at a novel negative response element of the 5-HT1A receptor gene. J    Biol Chem 276, 14299-14307.-   Packham, G., Brimmell, M., and Cleveland, J. L. (1997). Mammalian    cells express two differently localized BAG-1 isoforms generated by    alternative translation initiation. Biochem. J. 328, 807-813.-   Pichon, B and Christophe, D (1998) An in vitro transcription system    for the study of thyroid-specific transcription. Anal Biochem 261,    233-235.-   Pratt, W. B. and Dittmar, K. D. (1998). Studies with purified    chaperones advance the understanding of the mechanism of    glucocorticoid receptor-hsp90 heterocomplex assembly. Trends    Endocrinol Metabol 9, 244-252.-   Pratt, W. B. and Toft, D. O. (1997). Steroid receptor interactions    with heat shock protein and immunophilin chaperones. Endocr Rev 18,    306-360.-   Savory, J G, Prefontaine, G G, Lamprecht, C, Liao, M, Walther, R F,    Lefebvre, Y A, Hache, R J (2001) Glucocorticoid Receptor Homodimers    and Glucocorticoid-Mineralocorticoid Receptor Heterodimers Form in    the Cytoplasm through Alternative Dimerization Interfaces. Mol Cell    Biol 21, 781-793.-   Schenborn, E Groskreutz, D (1999) Reporter gene vectors and assays.    Mol Biotechnol 13, 29-44.-   Scheufler, C., Brinker, A., Bourenkov, G., Pegoraro, S., Moroder,    L., Bartunik, H., Hartl, F. U., and Moarefi, I. (2000). Structure of    TPR domain-peptide complexes: critical elements in the assembly of    the Hsp70-Hsp90 multichaperone machine. Cell 101, 199-210.-   Schneikert, J., Hübner, S., Langer, G., Petri, T., J{umlaut over    (aa)}ttelä, M., Reed, J., and Cato, A. C. (2000). Hsp70-RAP46    interaction in downregulation of DNA binding by glucocorticoid    receptor. EMBO J. 19, 6508-6516.-   Schneikert, J., Hübner, S., Martin, E., and Cato, A. C. (1999). A    nuclear action of the eukaryotic cochaperone RAP46 in downregulation    of glucocorticoid receptor activity. J Cell Biol 146, 929-940.-   Service, R. F. (2000). Biochemistry. Chemical tags speed delivery    into cells. Science 288, 28-29.-   Silverman, L, Campbell, R, Broach, J R (1998) New assay technologies    for high-throughput screening. Curr Opin Chem Biol 2, 397-403.-   Sondermann, H., Scheufler, C., Schneider, C., Höhfeld, J., Hartl, F.    U., and Moarefi, I. (2001). Structure of a Bag/Hsc70 Complex:    Convergent Functional Evolution of Hsp70 Nucleotide Exchange    Factors. Science 291, 1553-1557.-   Song, J., Takeda, M., and Morimoto, R. I. (2001). Bag1-Hsp70    mediates a physiological stress signalling pathway that regulates    Raf-1/ERK and cell growth. Nat. Cell Biol. 3, 276-282.-   Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J.,    Seeburg, P. H., and Journot, L. (1993). Differential signal    transduction by five splice variants of the PACAP receptor. Nature    365,170-175.-   Trapp, T Holsboer, F (1996) Heterodimerization between    mineralocorticoid and glucocorticoid receptors increases the    functional diversity of corticosteroid action. Trends Pharmacol Sci    17, 145-149.-   Takayama, S., Krajewski, S., Krajewska, M., Kitada, S., Zapata, J.    M., Kochel, K., Knee, D., Scudiero, D., Tudor, G., Miller, G. J.,    Miyashita, T., Yamada, M., and Reed, J. C. (1998). Expression and    location of Hsp70/Hsc-binding anti-apoptotic protein BAG-1 and its    variants in normal tissues and tumor cell lines. Cancer Res 58,    3116-3131.-   Takayama, S., Sato, T., Krajewski, S., Kochel, K., Irie, S.,    Millan, J. A., and Reed, J. C. (1995). Cloning and functional    analysis of BAG-1: a novel Bcl-2-binding protein with anti-cell    death activity. Cell 80, 279-284.-   Thress, K., Song, J., Morimoto, R. I., and Kornbluth, S. (2001).    Reversible inhibition of Hsp70 chaperone function by Scythe and    Reaper. EMBO J. 20, 1033-1041.-   Toft, D. O. (1998). Recent advances in the study of hsp90 structure    and mechanism of action. TEM 9, 238-243.-   Turner, B. C., Krajewski, S., Krajewska, M., Takayama, S., Gumbs, A.    A., Carter, D., Rebbeck, T. R., Haffty, B. G., and Reed, J. C.    (2001). BAG-1: a novel biomarker predicting long-term survival in    early-stage breast cancer. J. Clin. Oncol. 19, 992-1000.-   Witcher, M., Yang, X., Pater, A., and Tang, S. C. (2001). BAG-1 p50    Isoform Interacts with the Vitamin D Receptor and Its Cellular    Overexpression Inhibits the Vitamin D Pathway. Exp. Cell Res.    265,167-173.-   Yang, X., Chernenko, G., Hao, Y., Ding, Z., Pater, M. M., Pater, A.,    and Tang, S. C. (1998). Human BAG-1/RAP46 protein is generated as    four isoforms by alternative translation initiation and    overexpressed in cancer cells. Oncogene 17, 981-989.-   Zeiner, M., Gebauer, M., and Gehring, U. (1997). Mammalian protein    RAP46: an interaction partner and modulator of 70 kDa heat shock    proteins. EMBO J. 16, 5483-5490.-   Zeiner, M. and Gehring, U. (1995). A protein that interacts with    members of the nuclear hormone receptor family: identification and    cDNA cloning. Proc. Natl. Acad. Sci. U.S.A. 92, 11465-11469.-   Zeiner, M., Niyaz, Y., and Gehring, U. (1999). The hsp70-associating    protein Hap46 binds to DNA and stimulates transcription. Proc Natl    Acad Sci USA 96, 10194-10199.

1. A method of identifying a compound that inhibits or reduces bag-1mediated downregulation of glucocorticoid-receptor (GR) transactivationsaid method comprising the steps of (a) contacting a test compound or aplurality of test compounds with (aa) a (poly)peptide comprising themotif X-Lys-Lys-Lys-Y-Arg-Arg-Arg wherein X and Y represent 0, 1, 2, 3,4, 5 or 6 amino acids and wherein said amino acids allow binding of themotif to double-stranded DNA; and (ab) a double-stranded DNA underconditions that allow binding of said (poly)peptide to said DNA in theabsence of said test compound or said plurality of test compounds; and(b) assessing whether binding of said (poly)peptide to said DNA occursin the presence of said test compound or said plurality of testcompounds wherein inhibition or reduction of binding is indicative ofthe test compound(s) being suitable to inhibit or reduce bag-1 mediateddownregulation of GR transactivation.
 2. The method of claim 1 whereinsaid motif recited in (aa) is located at the N-terminus of said(poly)peptide.
 3. The method of claim 1 wherein said DNA carries areadout-system that is activated by the binding of said (poly)peptide tosaid DNA.
 4. A method of identifying a compound that inhibits or reducesbag-1 mediated downregulation of glucocorticoid-receptor transactivationsaid method comprising the steps of (a) contacting a test compound or aplurality of test compounds with (aa) a (poly)peptide comprising themotif X-Lys-Lys-Lys-Y-Arg-Arg-Arg preferably at its N-terminus wherein Xand Y represent 0, 1, 2, 3, 4, 5 or 6 amino acids; and a domain that isthe hsp70 binding domain of bag-1 or functionally equivalent thereto;(ab) a polypeptide representing the GR or a functionally equivalentmolecule; and (ac) a double-stranded DNA molecule comprising a bindingsite for the GR under conditions that allow the formation of afunctional (poly)peptide complex consisting of said (poly)peptidesrecited in (aa) to (ab) and binding of said polypeptide recited in (ab)with said double-stranded DNA molecule in the absence of said testcompound or said plurality of test compounds; and (ba) assessing whethersaid polypeptide recited in (ab) binds to said double-stranded DNAwherein an increase of binding is indicative of the test compound(s)being suitable to inhibit or reduce bag-1 mediated downregulation of GRtransactivation; or (bb) assessing whether said complex formation and/orDNA-binding recited in (ac) results in a transactivation of GR whereinan increased level of transactivation is indicative of the testcompound(s) being suitable to inhibit or reduce bag-1 mediateddownregulation of GR transactivation.
 5. The method of claim 4 whereinsaid test compound or plurality of test compounds, said (poly)peptidesrecited in steps (aa) and (ab) and said double-stranded DNA molecule arefurther contacted with (ad) a (poly)peptide comprising the bag-1 bindingdomain of hsp70 or a functionally equivalent domain and the GR bindingdomain of hsp70 or a functionally equivalent domain.
 6. The method ofclaim 4 or 5 wherein the amino acids X and Y comprised in the(poly)peptide recited in (aa) allow binding of the motif to adouble-stranded DNA.
 7. The method of claim 4 or 5 wherein said testcompound or plurality of test compounds is/are further contacted with adouble-stranded DNA molecule that does not comprise a binding site forGR.
 8. The method of claim 4 wherein said double-stranded DNA moleculerecited in (ac) further carries a readout-system that is activated uponbinding of said GR to said DNA.
 9. The method of claim 3 or 8 whereinsaid readout-system comprises a reporter gene.
 10. The method of claim 9wherein said reporter gene is selected from the group consisting offirefly luciferase, renilla luciferase, β-galactosidase, GFP or anotherfluorescent protein, CAT (chloramphenicolacetyltransferase), alkalinephosphotase including SEAP (secreted alkaline phosphatase), TAT (tyrosylaminotransferase) and peroxidase.
 11. The method of claim 9 wherein saidreporter gene is a gene encoding a transmembrane protein.
 12. The methodof claim 11 wherein said transmembrane protein is a receptorpolypeptide.
 13. The method of any one of claims 1 to 5 wherein saidtest compound(s) is/are selected from the group consisting of smallmolecules, peptides, aptamers and antibodies or fragments or derivativesthereof.
 14. The method of claim 13 wherein said small molecules aresmall organic molecules.
 15. The method of claim 13 wherein saidpeptides are derived from an at least partially randomized peptidelibrary.
 16. The method of claim 13 wherein said antibodies aremonoclonal antibodies.
 17. The method of any one of claims 1 to 5wherein, if a plurality of test compounds is tested, (a) differentmembers of said plurality of test compounds are tested in differentreaction vessels wherein those reaction vessels that do not contain testcompounds indicative of being suitable to inhibit or reduce bag-1mediated downregulation of GR transactivation are not furtherconsidered; (b) members contained in reaction vessels that test positivewith regard to inhibition or reduction of bag-1 mediated downregulationof GR transactivation are redistributed into different reaction vesseland tested again; and optionally (c) step (b) is repeated until a singlecompound is identified that is suitable to inhibit or reduce bag-1mediated downregulation of GR transactivation.
 18. The method of any ofclaims 1 to 5 wherein the assessment is effected in an in vitrotranscription/translation system or using bacteriophage based systemsusing the T7, T3 or Sp6 promoter.
 19. The method of any of claims 1 to 5wherein the assessment is effected in an eukaryotic cell or tissue or anextract thereof.
 20. The method of any one of claims 1 to 5 wherein X isMet.
 21. The method of any one of claims 1 to 5 wherein Y is Thr. 22.The method of any one of claims 1 to 5 wherein Y is
 0. 23. The method ofany one of claims 1 to 5 wherein Y is Ala-Thr.
 24. The method of any oneof claims 1 to 5 wherein Y is Ala-Ala-Thr.
 25. The method of any one ofclaims 1-5 wherein said (poly)peptide comprising the motifX-Lys-Lys-Lys-Y-Arg-Arg-Arg is bag-1.
 26. The method of claim 5 whereinsaid (poly)peptide recited in step (ad) is hsp70.
 27. The method ofclaim 1 or 5 further comprising refining the identified compound,comprising the steps of: (i) identification of the binding site of saidcompound binding to said motif and optionally of the binding site ofsaid motif binding to said compound; (ii) molecular modeling of thebinding site of the compound and optionally of the motif; and (iii)modification of the compound to improve its binding specificity for themotif.
 28. The method of claim 1 or 5 further comprising refining theidentified compound, comprising: (a) modeling said compound bypeptidomimetics; and (b) chemically synthesizing the modeled compound.29. The method of claim 1 or 5, further comprising modifying theidentified compound, comprising attaching said compound to a signalpeptide.
 30. The method of claim 1 or 5, further comprising modifyingthe identified compound as a lead compound to achieve (i) modified siteof action, spectrum of activity, organ specificity; and/or (ii) improvedpotency; and/or (iii) decreased toxicity (improved therapeutic index);and/or (iv) decreased side effects; and/or (v) modified onset oftherapeutic action, duration of effect; and/or (vi) modifiedpharmakinetic parameters (resorption, distribution, metabolism andexcretion); and/or (vii) modified physico-chemical parameters(solubility; hygroscopicity; color, taste, odor, stability, state);and/or (viii) improved general specificity, organ/tissue specificity;and/or (ix) optimized application form and route by (i) esterificationof carboxyl groups; or (ii) esterification of hydroxol groups withcarbon acids; or (iii) esterification of hydroxol groups to, e.g.phosphates, pyrophosphates or sulfates or hemi succinates; or (iv)formation of pharmaceutically acceptable salts; or (v) formation ofpharmaceutically acceptable complexes; or (vi) synthesis ofpharmacologically active polymers; or (vii) introduction of hydrophilicmoieties; or (viii) introduction/exchange of substituents on aromates orside chains, change of substituent pattern; or (ix) modification byintroduction of isoteric or bioisoteric moieties; or (x) synthesis ofhomologus compounds; or (xi) introduction of branched side chains; or(xii) conversion of alkyl substituents to cyclic analogues; or (xiii)derivatisation of hydroxyl group to kelates, acetates; or (xiv)N-acetylation to amides, pheycarbamates; or (xv) synthesis of Mannichbases; imines; or (xvi) transformation of ketones or aldehydes toSchiffs bases, oximes, acetates, ketales, enolesters, oxazolidines,thiozolidines; or combinations thereof.
 31. The method of claim 1 or 5,further comprising producing a pharmaceutical composition comprising thestep of formulating one or more of the identified compounds with apharmaceutically acceptable carrier or diluent.
 32. (canceled)
 33. Themethod of claim 29, wherein the identified compound has been refined bythe method of claim 27 or 28 before said compound is further modified.34. The method of claim 30, wherein the identified compound has beenrefined by the method of claim 27 or 28 before said compound is furthermodified.
 35. The method of claim 31, wherein the identified compoundhas been refined by the method of claim 27 or 28 before said compound.